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* These authors contributed equally
This protocol presents a step-by-step methodological approach of exposing bone metastatic cells lines and primary bone tumors to 5-aminolevulinic acid-mediated photodynamic therapy (PDT). The effects on cell migration potential/invasiveness, viability, apoptosis, and senescence potential are also analyzed following PDT exposure.
Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive therapy that can target local metastatic bone lesions. This paper presents an in vitro method to study the PDT effect in adherent cell lines. To this end, we demonstrate a step-by-step approach to subject both primary (giant cell bone tumor) and human bone metastatic cancer cell lines (derived from a primary invasive ductal breast carcinoma and renal carcinoma) to 5-aminolevulinic acid (5-ALA)-mediated PDT.
After 24 h post 5-ALA-PDT irradiation (blue light-wavelength 436 nm), the therapeutic effect was assessed in terms of cell migration potential, viability, apoptotic features, and cellular growth arrest (senescence). Post 5-ALA-PDT irradiation, musculoskeletal-derived cell lines respond differently to the same doses and exposure of PDT. Depending on the extent of cellular damage triggered by PDT exposure, two different cell fates-apoptosis and senescence were noted. Variable sensitivity to PDT therapy among different bone cancer cell lines provides useful information for selecting more appropriate PDT settings in clinical settings. This protocol is designed to exemplify the use of PDT in the context of musculoskeletal neoplastic cell lines. It may be adjusted to investigate the therapeutic effect of PDT on various cancer cell lines and various photosensitizers and light sources.
Therapeutic options for bone metastases are still limited and challenging despite ongoing developments in oncological treatment. The current standard method is radiotherapy, which is associated with complications such as local erythema, toxicity to inner organs1, and insufficient fractures2. There is a need for alternative antineoplastic therapies as patients with bone metastases often suffer from pain, hypercalcemia, and neurological symptoms that result in impaired mobility and reduced quality of life3. Recent findings demonstrate that PDT provides a promising, alternative, antineoplastic treatment option to directly target bone lesions, which can be used alone or supportively to radiotherapy4.
The mechanism of PDT is essentially based on an energy transfer from a light-excited photosensitive compound (photosensitizer) to tissue oxygen. This photosensitizer works similarly to a capacitor on a nanoscopic level. It can store energy in a ground state when irradiated with an appropriate wavelength of light and releases stored energy when it returns from an excited state to the original ground state5. The released energy leads to two photochemical reactions: one is the transformation of oxygen to reactive oxygen radicals by transferring hydrogen or an electron. The second is the production of singlet oxygen particles by horizontal energy transfer from the photosensitizer substrate to local triplet oxygen particles6. Reactive oxygen radicals and singlet oxygen molecules have highly cytotoxic effects on local tumor cells and induce vascular occlusion and local inflammatory response by apoptosis of endothelial cells of tumor blood vessels7.
Conventional photosensitizers are derivatives of the porphyrin family such as hematoporphyrins and benzoporphyrins8. Applying photosensitizer substances with higher affinity to tumor tissue can increase the selectivity of PDT9 y. In particular, 5-ALA, which is a biosynthetic precursor of protoporphyrin IX, can accumulate in tumor cells such as actinic keratosis, basal cell carcinoma, bladder tumor, and gastrointestinal cancer5. Different delivery approaches using 5-ALA can also vary the efficiency of PDT in relation to tumor localization. Thus, topical use of 5-ALA with the application of PDT became the first-line dermatologic therapy against actinic keratosis10. Recent results for bone metastases of invasive ductal breast cancer cell lines indicate possible inhibition of cell migration and induction of apoptosis after exposure to PDT with 5-ALA11. However, using PDT in subfascial human tissue such as bone tissue is still in its preclinical to experimental clinical stage as the efficacy needs to be improved. Applications of nanoparticles with light-based therapy already show great impact in dentistry12. Thus, it is likely that combining the use of nanoparticles with PDT will expand its application range towards orthopedic oncology.
The following protocol describes how to prepare both cells originating from primary bone tumors and bone metastases cell lines and subject them to 5-ALA-mediated PDT for a predefined time exposure. A detailed description of how to perform and assess the cellular migration potential, vitality, and senescence post 5-ALA-PDT irradiation is also included. Step-by-step instructions provide a straightforward and concise approach to acquire reliable and reproducible data. The advantages, limitations, and future perspectives of the PDT approach for bone neoplastic lesions are also discussed.
Three different types of cell lines were employed: "MAM"-a cell line originating from bone metastases of renal cell carcinoma, "MAC"-bone metastases of an invasive ductal breast carcinoma, and "17-1012"-a giant cell tumor of bone. Marrow-derived mesenchymal stem cells (MSCs) were used as a control group. Institutional and ethical approval was obtained before the commencement of the study (project number: 008/2014BO2-for the cancer cell lines and project number: 401/2013 BO2 for MSCs).
1. Cell culture
NOTE: Culture media can be prepared beforehand. The culture medium for MAM and 17-1012 consists of RPMI supplemented with 10% (v/v) fetal bovine serum (FBS) and 2 mM L-glutamine. The culture medium for MAC and MSCs consists of Dulbecco's modified Eagle's medium (DMEM) with glutamine substitute (see the Table of Materials), 4.5 g/L D-glucose supplemented with 10% (v/v) FBS.
2. PDT setup and exposure
3. Migration assay
4. Viability assay
5. Cellular growth arrest/senescence assay (β-Galactosidase( β-Gal) activity)
NOTE: All reagents and buffers used here were provided in the assay kit (see the Table of Materials).
Following 5-ALA PDT exposure, the MSC-control group showed no notable effect in terms of migration following 5-ALA PDT irradiation (Figure 2A, i, v, ix). In contrast, MAC cells (Figure 1B and Figure 2A, iii, vii, xi) and 17-1012 (Figure 1B and Figure 2A, ii, vi, x) cells exhibited a decrease in migration potential for both ...
Despite current treatment options, cancer therapeutic response is variable, advocating in favor of novel approaches or even combination therapies to treat bone metastases while preserving the initial tissue structure. In this context, PDT is a promising alternative. From a simplistic point of view, PDT is comprised of two basic components: (1) a nontoxic light-sensitive dye termed photosensitizer (PS) and (2) an external light source of the appropriate wavelength that matches the absorption spectrum of the PS and activat...
The authors have no conflicts of interest to disclose.
We thank our co-authors from the original publications for their help and support.
Name | Company | Catalog Number | Comments |
300 s metered card for PDT | IlluminOss Medical Inc., East Providence, Rhode Insland, USA | n/a | http://www.illuminoss.com |
5-aminolevulinic acid (5-ALA) photosensitizer | Sigma-Aldrich, St. Louis, Missouri, USA | A7793 | 10 mg |
6 Well plates | Greiner Bio-One, Frickenhausen, Germany | 657160 | |
8 Well Chamber Slides | SARSTEDT AG & Co. KG, Munich, Germany | 94.6140.802 | |
96 Well plates (F-buttom) | Greiner Bio-One, Frickenhausen, Germany | 655180 | |
CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS-Assay) | Promega, Fitchburg, Wisconsin, USA | G3580 | |
Cellular Senescence Assay | Biotrend Chemikalien GmbH, Köln, Germany | CBA-231 | Quantitative senescence-associated ß-galactosidase assay |
Coomassie Brilliant Blue R250 | Sigma-Aldrich, St Louis, Missouri, USA | 35055 | 0.5% (w/v) |
Culture-Inserts 2Well | ibidi GmbH, Gräfelfing, Germany | 80209 | |
DMEM (1x) + GlutaMax-I | Life Technologies, Carlsbad, Kalifornien, USA | 31966-021 | |
Fetal bovine serum (FBS) | Sigma-Aldrich, St Louis, Missouri, USA | F7524 | |
Fluorescence microplate reader | Promega, Madison, Wisconsin, USA | GlowMAx®, GM3510 | |
Hemocytometer | Hecht Assistent, Sondheim, Deutschland | 4042 | |
ImageJ | National Institutes of Health, Be-thesda, Maryland, USA | ImageJ (version: 1.53a) | Software for processing and analyzing scientific images; https://imagej.net/ |
Inverse phase-contrast microscope | Leica, Wetzlar, Germany | DM IMBRE 100 | |
Methanol AnulaR Normapur | VWR, Fontenay-Sous-Bois, France | 20847.307 | |
Paraformaldehyd | Sigma-Aldrich, St Louis, Missouri, USA | 158127 | Powder, 95% purity |
PDT device (light box and accesories) | IlluminOss Medical Inc., East Providence, Rhode Insland, USA | n/a | Blue light 436 nm, 36 J/cm2 http://www.illuminoss.com |
Penicillin-Streptomycin | Thermo Fisher Scientific, Waltham, Massachusetts, USA | 15140-122 | 10,000 U/mL Penicillin 10,000 μg/mL Streptomycin |
Phosphate-buffered saline (PBS) | Thermo Fisher Scientific, Waltham, Massachusetts, USA | 10010-015 | |
RPMI 1640 | Thermo Fisher Scientific, Waltham, Massachusetts, USA | 21875034 | |
Spectrophotomete/ microplate reader | BioTek Instruments GmbH, Bad Friedrichshall, Germany | EL800 | |
Trypan Blue dye 0.4% | Sigma-Aldrich, St Louis, Missouri, USA | T8154 | |
Trypsin-EDTA 10x | Sigma-Aldrich, St Louis, Missouri, USA | T4174 |
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